This invention relates to a method for selectively amplifying target RNA relative to non-target RNA in a sample of tester RNA.
Disease is a deviation from the normal functioning of the body""s organs or systems. This deviation can arise in a number of ways: by either an abnormal gene being switched on or by a normal gene being switched off; by chromosomal mutations or rearrangements which frequently result in abnormal or missing gene products in congenital conditions; or by the presence of an infectious agent in genetically normal individuals. In some cases, the total complement of mRNA, the products of gene expression, in an abnormal cell Will be different from that in a normal cell. In other cases, there may be no apparent difference in the level of gene expression, but the genetic lesion may be a subtle point mutation giving rise to a defective gene product.
Identification of differences in genetic expression or sequence between normal and abnormal cells is a powerful diagnostic and/or prognostic tool. It can also be the first step in understanding a disease by revealing its underlying mechanism. Thus, identification of genetic differences between normal and abnormal cells can provide a clear path to the design of new diagnostic tests, new drugs or gene therapy.
Methods for identifying and isolating sequences present or actively expressed in one cell but diminished or absent in another cell, referred to as xe2x80x9cdifferential screeningxe2x80x9d and xe2x80x9cdifference cloningxe2x80x9d, have been applied to both genomic DNA and mRNA. Difference cloning is based upon subtractive hybridization, a method for isolating xe2x80x9ctargetxe2x80x9d sequences from one DNA population, referred to in this application as xe2x80x9ctesterxe2x80x9d, by using an excess of sequences from another DNA population, referred to in this application as xe2x80x9cdriverxe2x80x9d. One method of subtractive hybridization mixes a restriction endonuclease-digested tester DNA with an excess of randomly sheared driver DNA Lamar, et al., xe2x80x9cY-Encoded, Species-Specific DNA in Mice: Evidence That the Y Chromosome Exists in Two Polymorphic Forms in Inbred Strainsxe2x80x9d, Cell, Vol. 37, pp. 171-177, (1984). The DNA mixture is denatured, hybridized and ligated into a compatible restriction site in a cloning vector. Only a tester DNA fragment reannealled to its complement would have both of the correct ends required for cloning. Conversely, any tester DNA fragments that anneal to complementary driver DNA fragments would not have both of the required ends. The low yield of cloned target sequences in this method is due primarily to the slow reannealling of dilute tester sequences to their complements. In addition, the enrichment of unique target sequences from a background of sequences common to the driver is limited by the initial excess of driver to tester.
Other methods of subtractive hybridization are directed toward the preparation of subtracted probes for differential screening of cDNA libraries by in situ colony blot hybridization. In differential screening, differentially expressed nucleic acids are not cloned, but are used as hybridization probes to identify and characterize unenriched cDNA clones. One method described by Kuze, et al., xe2x80x9cA new vector and RNase H method for the subtractive hybridizationxe2x80x9d, Nucleic Acids Research, Vol. 17, No. 2, pp. 807, (1989). prepares subtracted RNA probes using hybridization to DNA, followed by digestion with RNase H to separate non-hybridized RNA from the hybrid. After the remaining RNA is purified, the subtractive hybridization process is repeated. Hybridization of immobilized DNA using the purified subtracted RNA probe indicates that the subtracted probes can be enriched at least 100 fold. Kuze et al. does not describe a way in which the sequences of the subtracted RNA itself may be cloned or amplified, or suggest a use for the subtracted RNA other than as a hybridization probe.
Some improvements to the subtractive hybridization methods, as applied to difference cloning, involve the use of nucleic acid amplification processes selectively to increase the copy number of a DNA segment having the target sequence. An improvement to subtractive hybridization described by Wieland, et al., xe2x80x9cA method for difference cloning: Gene amplification following subtractive hybridizationxe2x80x9d, Proc. Natl., Acad. Sci. USA, Vol. 87, pp. 2720-2724, (1990). Uses the xe2x80x9cpolymerase chain reactionxe2x80x9d (PCR) to increase the concentration of target sequences, and multiple steps of annealing tester DNA to excess driver DNA to further enrich the unique target sequences from a background of sequences common to the driver. in this procedure tester DNA fragments are first prepared for amplification by ligating to a xe2x80x9ctemplatexe2x80x9d oligonucleotide. A mixture of prepared tester DNA and a 200-fold excess of randomly sheared driver DNA is denatured and reannealled to 90% completion, after which the remaining single-stranded DNA containing target sequences is purified from the double-stranded DNA containing the driver. After three rounds of denaturation, annealing and purification, the remaining tester DNA is then amplified in PCR using primers that anneal to the template sequences. The double-stranded PCR products are then cloned and sequenced. The method gave a 100- to 700-fold enrichment of target sequences.
Another improvement to subtractive hybridization described by Lisitsyn, et al., xe2x80x9cCloning the Differences Between Two Complex Gnomesxe2x80x9d, Science, pp. 946-951, (1993). is a technique called xe2x80x9crepresentational difference analysisxe2x80x9d (RDA). RDA lowers the complexity of both tester and driver genomic DNA by using various restriction endonucleases, eg. BamHI, Bg/II and HindIII, to generate fragments of a particular length that can be efficiently amplified in PCR as xe2x80x9crepresentationsxe2x80x9d of the genome. The tester and driver fragments are ligated to dephosphorylated oligonucleotide adaptors such that an adaptor sequence is ligated to the 5xe2x80x2-end of each strand. The adapted fragments are then amplified in separate PCR reactions using the adaptor as a primer to achieve kinetic enrichment of a population of xe2x80x9campliconsxe2x80x9d that are below 1 kb in size. Finally, the tester and driver amplicons is digested with the same restriction endonuclease to remove the original adaptors.
The xe2x80x9cdifference analysisxe2x80x9d step of RDA is based upon the kinetic and subtractive enrichment of the tester amplicons in a second PCR. It begins with ligating different dephosphorylated oligonucleotide adaptors, this time only to the tester amplicons. An excess of driver amplicons are then mixed with the adapted tester fragments, denatured, and allowed to anneal. A portion of the annealed fragments is then treated with a DNA polymerase to allow extension of driver and adapted tester strands using the complementary driver or adapted tester strands as template. The annealed and extended amplicons are then amplified in PCR using the adaptor as a primer. Driver strands annealed to complementary driver strands will not be extended or amplified. Driver strands annealed to complementary adapted tester strands will be extended, but will lack a 5xe2x80x2-terminal adaptor sequence that is necessary to form a template for exponential amplification in PCR. Only the adapted tester strands that anneal to their complementary adapted tester strands and are extended prior to PCR will contain sequences on both 5xe2x80x2 and 3xe2x80x2 ends to enable exponential amplification.
Following 10 cycles of PCR, a portion of the amplified products is treated with a nuclease to specifically degrade single-stranded nucleic acids. After inactivating the nuclease, a portion of the remaining nucleic acids is further amplified in PCR using the same primer. After 15 to 20 more cycles of PCR, the double-stranded DNA products are digested with the restriction endonuclease, and the process of difference analysis is repeated. After sufficient rounds (typically 3 to 4, for example) of RDA are performed, the double-stranded DNA products are finally cloned and analyzed.
Although RDA was originally developed for genomic DNA, a variation of RDA has been more recently applied to cDNA Hubank, et al., xe2x80x9cIdentifying differences in mRNA expression by representational difference analysis of cDNAxe2x80x9d, Nucleic Acids Research, Vol. 22, No. 25, pp. 5640-5648, (1994). Since the cDNA population derived from a typical cell represents only 1-2% of the total genome, kinetic enrichment to reduce complexity should not be necessary for RDA when applied to cDNA. Dephosphorylated oligonucleotide adaptors are ligated to tester and driver cDNA fragments that are generated by digestion of the double-stranded cDNA with a frequently cutting restriction endonuclease, eg. DpnII. The adapted cDNA fragments are amplified separately by PCR and the resulting amplicons are digested with the same restriction endonuclease to remove the adaptor sequences. The tester cDNA amplicons are then subjected to multiple rounds of RDA as described for genomic DNA. Despite many successful applications of subtractive hybridization, the various methodologies have been described as xe2x80x9ctechnically difficult, time consuming and often either impractical or unreliablexe2x80x9d (Hubank and Schatz, 1994). One difficulty encountered with subtractive hybridization is the requirement for physical removal of hybridized tester sequences prior to amplification and/or cloning. Another limitation of subtractive hybridization is related to self-reassociation kinetics of complex genomes. Similarly, RDA shares some of the problems common to other subtractive hybridization methods. One is the requirement for self-reannealling of complementary tester sequences to enable amplification. For rare cDNA sequences (less than 1%, for example) this imposes kinetic limitations and requires lengthy hybridizations (of 20 hours, for example). Another limitation of RDA is the amplification of a double-stranded tester DNA that requires the use of an excess (typically 100-fold) of double-stranded driver DNA to compete with the re-annealing of complementary tester DNA strands. A single-stranded tester nucleic acid could more effectively hybridize with a complementary single-stranded driver DNA. To avoid physical removal of hybridized sequences, RDA requires the covalent activation and inactivation of tester sequences prior to amplification. [For subsequent rounds of RDA, these modifications must be removed with a restriction enzyme, new primers must be ligated and the newly modified tester must be subjected to hybridization with driver and activation/inactivation steps before the next application of PCR.] This multi-step process becomes a serious practical problem with RDA, in that the PCR amplification step is vulnerable to contamination by less enriched amplified products. Anti-contamination procedures cannot be implemented since the amplicons must remain intact and active as a template throughout the various steps of each round of RDA.
Thus, there is a need for a process for the enrichment of nucleic acid sequences which: 1) avoids hybrid removal prior to amplification; 2) does not require concentration-limiting self-annealing of tester nucleic acids; 3) utilizes single-stranded tester and driver nucleic acids; 4) avoids modification of the tester nucleic acids between rounds of subtractive hybridization and amplification; and 5) integrates hybridization, inactivation, and exponential amplification into a process with fewer steps, thus avoiding contamination.
The method of this invention, called subtractive amplification, represents a novel combination of subtractive hybridization and nucleic acid amplification. Subtractive amplification has the following advantages: 1) it avoids the physical removal of hybridized sequences before amplification; 2) it avoids a concentration-limiting self-annealing of tester nucleic acids in order to activate them for amplification; 3) it improves hybridization efficiency by using single-stranded tester and driver nucleic acids; 4) it avoids modification of the tester nucleic acids after the hybridization step to activate them for amplification; 5) it avoids modification of the tester nucleic acids between rounds of subtractive hybridization and amplification; 6) it allows a simple dilution procedure to prepare the products of one round of subtractive amplification reaction for the next round of subtractive amplification; and 7) it minimizes contamination by integrating multiple rounds of subtractive hybridization and exponential amplification into a process with fewer steps.
According to the method of the present invention, target RNA is preferentially amplified relative to non-target RNA in a sample of tester RNA. As used herein, tester RNA refers to RNA sequences which are to be subjected to subtractive amplification according to the present invention. Tester RNA includes both target and non-target RNA sequences, each containing terminal priming sequences to enable amplification. In the method, target RNA is preferentially amplified relative to any amplification of non-target RNA that may occur.
According to the method, a sample of tester RNA is contacted with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA. A nucleic acid primer is then extended using the target RNA as a template, forming a DNA template complementary to some or all of the target RNA. The DNA template is then rendered single-stranded to enable the hybridization to it of a promoter template, and extended using the promoter template to form a DNA template comprising a functional double-stranded promoter. Multiple copies of the target RNA sequence can now be transcribed from the DNA template through recognition of the contained functional double-stranded promoter by RNA polymerase.
According to the method, the driver sequences, nucleic acid primer, and promoter template may be DNA or RNA, and are preferably DNA.
According to the method, the hybridization of driver sequences to the non-target RNA interferes with the ability of the non-target RNA to serve as a template. As a result, the nucleic acid primer is preferentially extended using the target RNA as a template for forming the DNA template, as opposed to using the non-target RNA as template. In one embodiment of the method, non-target RNA is inactivated as a template for DNA synthesis prior to the formation of the DNA template. Inactivation of the non-target RNA may be performed by a variety of methods including, for example, digesting any non-target RNA which is hybridized to a driver sequence. Digestion of non-target RNA may be performed, for example, by any enzyme having ribonucleolytic activity analogous to that of ribonuclease H enzyme.
After its formation, the DNA template is preferably rendered single-stranded. This may be accomplished by digesting any target RNA hybridized to the DNA template, for example, using any enzyme having ribonucleolytic activity analogous to that of ribonuclease H enzyme. The DNA template may also be rendered single-stranded through strand separation, for example, by heat denaturation.
The promoter template includes a sequence capable of forming a functional promoter. In one embodiment, the promoter template comprises sequences capable of forming a functional promoter and a transcription initiation site from a bacteriophage. In a preferred embodiment, the bacteriophage is T7.
According to the present invention, the DNA template and/or the extended DNA template can be amplified prior to target RNA synthesis. This amplification can be performed by a variety of nucleic acid amplification methods known in the art and is preferably performed either by the PCR (polymerase chain reaction) or SDA (strand displacement amplification) or NASBA (nucleic acid sequence-based amplification) methodologies.
In one embodiment of the present invention, the synthetic target RNA is synthesized from the extended DNA complement, the single-stranded DNA template containing a functional double-stranded promoter, by transcription utilizing RNA polymerase. In a further embodiment, the synthetic target RNA so transcribed may be further amplified by a RNA amplification reaction, preferably by NASBA.
Subtractive amplification can be performed in multiple cycles where the sample of tester RNA being used in the subtractive amplification process is synthetic target RNA obtained from a prior subtractive amplification. Driver sequences for a subsequent cycle of subtractive amplification can also be obtained from a prior subtractive amplification.
In one particular embodiment, subtractive amplification is performed by:
a) contacting the sample of tester RNA with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA;
b) contacting the target RNA with nucleic acid primers under conditions suitable for hybridization;
c) extending those nucleic acid primers which hybridize to the target RNA to form a DNA template complementary to some or all of the target RNA;
d) making the DNA template single-stranded;
e) contacting, under hybridizing conditions, the single-stranded DNA template with a nucleic acid sequence capable of acting as a promoter template;
f) extending those DNA templates which hybridize to the promoter template to form extended DNA templates comprising both a functional double-stranded promoter portion and a single-stranded portion capable of acting as a template; and
g) transcribing the extended DNA template to form multiple copies of synthetic target RNA.
According to a preferred embodiment of the method, target RNA present in a sample of tester RNA is preferentially amplified relative to non-target RNA present in the tester, both target and non-target RNA containing terminal priming sequences to enable amplification, by performing the following reactions: (1) a hybridization reaction, wherein the sample of tester RNA-contacts driver sequences under conditions such that the driver sequences which are complementary to the non-target RNA hybridize, and prevent the hybridized non-target RNA from functioning as templates for nucleic acid synthesis; (2) a reverse transcription reaction, wherein nucleic acid primers hybridize to the target RNA from the hybridization reaction and are extended using a reverse transcriptase, thereby forming a DNA template; (3) a DNA conversion reaction, wherein DNA templates from the reverse transcription reaction are made single-stranded, hybridized to a promoter template, and extended using the DNA polymerase, thereby forming DNA templates with a functional double stranded promoter; and (4) a transcription reaction, wherein DNA templates with functional double stranded promoters from the DNA conversion reaction are transcribed using an RNA polymerase, thereby forming from each DNA template one or more copies of the target RNA.
According to one aspect of the preferred embodiment, in each step of the process all or a portion of one reaction may be added to a subsequent reaction. In another aspect of the preferred embodiment, the hybridization reaction is performed by adding the tester RNA to a medium comprising driver sequences under conditions such that the driver sequences hybridize with non-target RNA. In another aspect of the preferred embodiment, the reverse transcription reaction is performed by adding a portion of the hybridization reaction to a medium comprising a first primer and a reverse transcriptase under conditions such that DNA templates are formed. In another aspect of the preferred embodiment, the DNA conversion reaction is performed by adding a portion of the reverse transcription reaction to a medium comprising a DNA polymerase and a promoter template under conditions such that DNA templates with functional double-stranded promoters are produced. In another aspect of the preferred embodiment, the transcription reaction is performed by adding a portion of the DNA conversion reaction to a medium comprising an RNA polymerase under conditions such that target RNA is produced.
According to one embodiment of the process, the driver nucleic acid sequences are composed of DNA. In one aspect of this embodiment, a portion of the hybridization reaction is added to a degradation reaction medium comprising a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences that anneal to the DNA driver sequences (non-target RNA) are degraded, and a portion of the degradation reaction is added to the reverse transcriptase reaction, hence providing target RNA sequences. In another aspect of this embodiment, the hybridization reaction medium further comprises a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences that anneal to the DNA driver sequences (non-target RNA) are degraded. Another aspect of this embodiment relates to the particular ribonucleases that may be used.
According to another embodiment of the process, a portion of the reverse transcription reaction is added to a degradation reaction medium comprising a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences forming a hybrid with the DNA templates are degraded, and a portion of the degradation reaction is added to the DNA conversion reaction, hence providing DNA templates.
According to another embodiment of the process, a portion of the reverse transcription reaction is added to a denaturation reaction under conditions such that the DNA templates are separated from the RNA tester sequences of the RNA:DNA hybrid, and a portion of the denaturation reaction is added to the DNA conversion reaction, hence providing DNA templates.
According to another embodiment of the process, the reverse transcription reaction medium further comprises a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences forming a hybrid with the DNA template are degraded.
According to another embodiment of the process, a portion of the reverse transcription reaction is added to a DNA amplification reaction medium comprising the first primer, a second primer and a DNA polymerase under conditions such that the DNA template hybridizes to the second primer and is extended using the DNA polymerase to form a double-stranded DNA and the strands of the double-stranded DNA are separated, upon which a cycle ensues wherein: i) the first primer and the second primer each hybridize to their complementary DNA strands; ii) each primer is then extended using the DNA polymerase to form a double-stranded DNA; and iii) the complementary DNA strands of the double-stranded DNA are separated, and thereafter a portion of the DNA amplification reaction is added to the DNA conversion reaction, hence providing DNA templates.
In one aspect of this embodiment, the DNA amplification reaction is the polymerase chain reaction, wherein the complementary DNA strands of the double-stranded DNA are separated by adjusting the reaction conditions to cause denaturation. In another aspect of this embodiment, the DNA amplification reaction is by strand displacement amplification, wherein the reaction medium further comprises a restriction endonuclease under conditions that the restriction endonuclease nicks the primers of the double-stranded DNA and the complementary DNA strands of the double-stranded DNA are separated by the DNA polymerase extending the nicked primers to displace the complementary DNA strands.
According to another embodiment of the process, the DNA conversion reaction medium further comprises a first primer, and the promoter template therein further comprises a second primer under conditions such that the DNA template hybridizes to the second primer and is extended using the DNA polymerase to form a double-stranded DNA, upon which a cycle ensues wherein: i) the complementary DNA strands of the double-stranded DNA are separated; ii) the first primer and the second primer each hybridize to their complementary DNA strands; and iii) each primer is then extended using the DNA polymerase to form a double-stranded DNA, thereby forming DNA templates with functional double-stranded promoters.
In one aspect of this embodiment, the complementary DNA strands of the double-stranded DNA are separated by adjusting the reaction conditions to cause denaturation. In another aspect of this embodiment, the reaction medium further comprises a restriction endonuclease under conditions that the restriction endonuclease nicks the primers of the double-stranded DNA and the complementary DNA strands of the double-stranded DNA are separated by the DNA polymerase extending the nicked primers to displace the complementary DNA strands.
According to another embodiment of the process, the reverse transcription, DNA conversion and transcription reactions together comprise an RNA amplification reaction, wherein tester RNA sequences are provided in a medium comprising a first primer, a promoter template, a reverse transcriptase, a DNA polymerase and an RNA polymerase under conditions such that the tester RNA sequences hybridize to the first primer and provide templates for synthesis of DNA templates by extension of the annealed first primer using the reverse transcriptase, thereby forming RNA:DNA hybrids, the RNA strands of which are degraded; the DNA templates hybridize to the promoter template and are extended using the DNA polymerase, thereby forming DNA templates each with a functional double-stranded promoter; and the RNA polymerase recognizes each double-stranded promoter and synthesizes from the DNA templates copies of the target RNA sequences.
In one aspect of this embodiment, the DNA polymerase is reverse transcriptase. In another aspect of this embodiment, the RNA amplification reaction medium further comprises a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences forming hybrids with the DNA templates are degraded. In another aspect of this embodiment, a portion of the hybridization reaction comprising the tester RNA sequences that do not anneal to the driver sequences is added to the RNA amplification reaction, hence providing tester RNA sequences. In another aspect of this embodiment, target RNA sequences from one round of the RNA amplification reaction provide tester RNA sequences in a subsequent round of the RNA amplification reaction. In another aspect of this embodiment, a portion of the RNA amplification reaction comprising target RNA sequences from one round of the process is added to the hybridization reaction of a subsequent round of the process, hence providing tester RNA sequences.
According to another embodiment of the process, target RNA sequences from one round of the process provide tester RNA sequences in a subsequent round of the process. In one aspect of this embodiment, a portion of the transcription reaction comprising target RNA sequences from one round of the process is added to the hybridization reaction of a subsequent round of the process, hence providing tester RNA sequences.
According to another embodiment of the process, a mixture of RNA comprising target RNA sequences and each comprising terminal priming sequences are added to the hybridization reaction, hence providing tester RNA sequences.
According to another embodiment of the process, the hybridization reaction further comprises an RNA polymerase, and to which is added a mixture of DNA templates each with a functional double-stranded promoter, under conditions such that the RNA polymerase recognizes each double-stranded promoter and synthesizes from the DNA templates copies of a mixture of RNA comprising target RNA sequences and each comprising terminal priming sequences, hence providing tester RNA sequences. In one aspect of this embodiment, terminal sequences and a double-stranded promoter are appended to DNA templates from which tester RNA sequences are synthesized using an RNA polymerase.
According to another embodiment, a final round of the subtractive amplification process comprises: 1) a hybridization reaction; 2) a reverse transcription reaction; 3) a DNA duplexing reaction, wherein DNA templates from the reverse transcription reaction are provided in a medium comprising a DNA polymerase and a second primer under conditions such that the DNA templates hybridize to the second primer and provide templates for synthesis of double-stranded DNA templates by extension of the annealed second primer using the DNA polymerase.
According to one aspect of this embodiment, the DNA duplexing reaction is performed within a DNA amplification reaction wherein the DNA duplexing reaction medium further comprises a first primer under conditions such that the second primer hybridizes to the DNA templates and is extended using the DNA polymerase to form double-stranded DNA templates, upon which a cycle ensues wherein: i) the complementary DNA strands of each double-stranded DNA template is separated; ii) the first primer and the second primer each hybridizes to their complementary DNA template strands; and iii) each primer is then extended using the DNA polymerase to form double-stranded DNA templates.
According to another aspect of this embodiment, the double-stranded DNA templates of the duplexing reaction are characterized by cloning and sequencing. In one aspect of this embodiment, the characterized sequences are used to make hybridization probes or amplification primers. In another aspect of this embodiment, the characterized sequences are used to identify or characterize a useful nucleic acid sequence. In another aspect of this embodiment, the cloned DNA templates are used for the preparation of tester RNA sequences or driver nucleic acid sequences.
The present invention also relates to kits for performing subtractive amplification according to the present invention. In general, these kits may include any compilation of RNA and DNA sequences and enzymes used to perform subtractive amplification according to the method of the present invention. In one embodiment, the kit includes a nucleic acid primer capable of hybridizing to a target RNA under conditions suitable for extension of the nucleic acid primer, and a promoter template capable of hybridizing to a DNA complement of the target RNA under conditions suitable for extension of the DNA complement such that a functional promoter is formed which is capable of transcribing the DNA complement such that multiple copies of target RNA are synthesized. In this kit, the nucleic acid primer and promoter template are preferably DNA. The promoter template may include sequences capable of forming a promoter and transcription initiation site from a bacteriophage. One type of bacteriophage may be T7. The kit may also include one or more enzymes which are used to perform one or more of the steps of subtractive amplification according to the present invention. Examples of these enzymes include: reverse transcriptase; a ribonuclease capable of digesting non-target RNA hybridized to a driver sequence, such as an enzyme having the ribonucleolytic activity of ribonuclease H; a RNA polymerase; and a DNA polymerase.